Carcinogenic Ability of Schistosoma Haematobium Possibly through ...
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Advances in Cancer: Research & Treatment
http://www.ibimapublishing.com/journals/ACRT/acrt.html
Vol. 2013 (2013), Article ID 876585, 8 pages
DOI: 10.5171/2013.876585
_____________
Cite this Article as: Mónica C Botelho, Isabel Veiga, Paula A Oliveira, Carlos Lopes, Manuel Teixeira, José M
Correia da Costa and José C Machado (2013), "Carcinogenic Ability of Schistosoma Haematobium Possibly
through Oncogenic Mutation of KRAS Gene," Advances in Cancer: Research & Treatment, Vol. 2013 (2013),
Article ID 876585, DOI: 10.5171/2013.876585
Research Article Carcinogenic Ability of Schistosoma
Haematobium Possibly through Oncogenic
Mutation of KRAS Gene
Mónica C Botelho1,2
, Isabel Veiga3, Paula A Oliveira
4, Carlos Lopes
5,6, Manuel
Teixeira3,5
, José M Correia da Costa1 and José C Machado
2,7
1INSA – National Institute of Health, Department of Health Promotion, Porto, Portugal
2IPATIMUP – Institute of Pathology and Molecular Immunology of Porto University, Porto, Portugal
3IPO - Portuguese Institute of Oncology, Department of Genetics, Porto, Portugal
4CECAV - Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD),
Vila Real, Portugal
5ICBAS - Institute of Biomedical Sciences Abel Salazar, Department of Cellular Biology and
Immunology, Porto University, Porto, Portugal
6IPO - Portuguese Institute of Oncology, Department of Pathology, Porto, Portugal
7FMUP - Faculty of Medicine of Porto University, Porto, Portugal
Received 9 January 2013; Accepted 16 January 2013; Published 28 April 2013
Academic Editor: Marta Herreros Villanueva
Copyright © 2013 Mónica C Botelho, Isabel Veiga, Paula A Oliveira, Carlos Lopes, Manuel Teixeira,
José M Correia da Costa and José C Machado. Distributed under Creative Commons CC-BY 3.0
Abstract
Schistosoma haematobium is a parasitic flatworm that infects millions of people, mostly in the
developing world, and is associated with high incidence of bladder cancer, although why is not
clear. Previously, we have used CD-1 mice to show that Schistosoma haematobium total antigen
(Sh) has a carcinogenic ability. Sh intravesically instillation induced the development of several
urothelial lesions, namely nodular hyperplasia and dysplasia (LGIUN—Low Grade Intra-
Urothelial Neoplasia) after 40 weeks of treatment. These results suggested that Sh induce
urothelium malignization. Bladder carcinoma frequently harbours gene mutations that
constitutively activate the receptor tyrosine kinase-Ras pathway for this reason we studied
activating mutations in KRAS gene. Twenty percent of the bladders with dysplasia presented a
KRAS mutation in codon 12 of exon 2. We concluded from these results that the parasite extract
of S. haematobium has carcinogenic ability possibly through oncogenic mutation of KRAS gene.
Keywords: Schistosoma haematobium, bladder cancer, oncogenes, kras mutation.
Advances in Cancer: Research & Treatment 2
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Mónica C Botelho, Isabel Veiga, Paula A Oliveira, Carlos Lopes, Manuel Teixeira, José M Correia da Costa and
José C Machado (2013), Advances in Cancer: Research & Treatment, DOI: 10.5171/2013.876585
Introduction
Schistosomiasis is a common parasitic
disease that ranks second only to malaria as
a global cause of morbidity and mortality. It
occurs in 76 tropical and subtropical
countries and affects about 10% of the
world’s population. The prevalence of this
infection remains high despite the
availability of efficacious antischistosomal
drugs. Individuals become infected with the
parasite at an early age by exposure to the
free-swimming larval stage (termed
cercariae) in contaminated water (Rosin et
al, 1994). Schistosomiasis is the first in
prevalence among all water-borne
infectious diseases and one of the main
occupational risks encountered in the rural
endemic areas of the world.
Schistosomiasis-associated bladder cancer
defines a characteristic pathological and
histogenic nature that differentiates it from
bladder cancer of non-schistosomal origin.
It shows an increased incidence of
squamous cell carcinoma as opposed to
transitional cell carcinoma, a lower mean
age at diagnosis, and a higher male/female
ratio. Moreover, in association with
schistosomiasis, cancer may originate
throughout the bladder but rarely in the
trigone, a frequent site of tumor origin in
non-schistosomal cases (Badawi, 1996).
Human bladder cancer is the fifth to the
seventh most common cancer in Western
countries. In many tropical and subtropical
areas, however, it is the first among all types
of cancer, mainly due to the endemic
parasitism (Badawi, 1996).
Many oncogenes participate in the
development of human tumors. The most
frequently activated genes are members of
the RAS gene family, two of which (Harvey-
H) and (Kirsten-K) were first identified as
being homologous to viral transforming
genes, while the third (N-ras) was identified
in a human neuroblastoma. The RAS gene
product is a monomeric membrane-
localized G protein of 21 Kd that functions
as a molecular switch linking receptor and
nonreceptor tyrosine kinase activation to
downstream cytoplasmic or nuclear events.
Each mammalian cell contains at least 3
distinct RAS proto-oncogenes encoding
closely related, but distinct proteins. RAS
genes can be activated by point mutation at
codons 12, 13, 61, or 113-117. Signal
transduction by Ras proteins involves
hydrolysis of GTP, and activating mutations
inhibit this process, locking the protein in
the “on” signaling conformation (Stanley,
1995).
Activating mutations in these Ras proteins
result in constitutive signaling, thereby
stimulating cell proliferation and inhibiting
apoptosis. Oncogenic mutations in the RAS
genes are present in approximately 30% of
all human cancers (Adjei, 2001). The high
degree of similarities shared by different
Ras isoforms suggests that functional
redundancy exists among these proteins, yet
accumulating data support for unique roles
of the Ras family members with K-ras being
the most important Ras isoform (Zhang et
al, 2007). Oncogenic KRAS mutations are
prevalent in virtually all cancer types,
making KRAS one of the most frequently
mutated genes in human cancers (Zhang et
al, 2007).
Mutational activation of RAS
protooncogenes is frequently observed in
human tumors and in experimental animals
(Cerutti P et al, 1994). KRAS mutations have
been found in bladder cancer patients (Jebar
et al, 2005; Gormally et al, 2006), in
transformed bladder epithelial cells
(Brookes et al, 1988) and chemically-
induced rat bladder tumors (Stanley, 1995).
We have previously demonstrated that
normal CHO (Chinese Hamster Ovary) cells
treated in laboratory with S. haematobium
total antigen, acquire cancer-like
characteristics. In fact, these cells presented
rapid uncontrolled division, high resistance
to death and an abnormal capability to
migrate (Botelho et al, 2009a) and, when
injected into nude mice, lead to tumour
development. Crucially, animals injected
with non-exposed cells showed no growth
(Botelho et al, 2009b). We have further used
CD-1 mice to show that Schistosoma
haematobium total antigen (Sh) has a
carcinogenic ability. Sh intravesically
instillation induced the development of
dysplasia in 70% of animals sacrificed 40
weeks after. Our results suggest that Sh
induce the malignization of the urothelium
(Botelho et al, 2011). Dysplasia (LGIUN—
Low Grade Intra-Urothelial Neoplasia) is
3 Advances in Cancer: Research & Treatment
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Mónica C Botelho, Isabel Veiga, Paula A Oliveira, Carlos Lopes, Manuel Teixeira, José M Correia da Costa and
José C Machado (2013), Advances in Cancer: Research & Treatment, DOI: 10.5171/2013.876585
preneoplastic lesion that can evolutes to
carcinoma in situ, invasive carcinoma
and/or papillary carcinoma. In the present
study, we addressed gene mutations that
constitutively activate the receptor tyrosine
kinase-Ras pathway and therefore we
studied activating mutations in KRAS gene.
Twenty percent of the bladders with
dysplasia presented a KRAS mutation in
codon 12 of exon 2. We concluded from
these results that the parasite extract of S.
haematobium may have carcinogenic ability
possibly through oncogenic mutation of
KRAS gene.
An improved understanding of the
molecular basis underlying bladder tumor
pathways and specifically schistosomiasis-
associated bladder cancer is of clear
importance in helping to better predict
bladder tumor progression and devise
optimal management strategies for this type
of carcinoma.
Material and Methods
Animals
Eight-week-old female golden hamsters
(LVG/SYR) and CD-1 mice were provided by
Charles River (Barcelona, Spain). Animals
spent one week being acclimated under
routine laboratory conditions before
starting the experiments. They did not
receive any treatment prior to the study.
Hamsters were kept in separated cages and
mice were kept in 6 litter mates cage. They
were fed standard balanced food and water
ad libitum. All the animals were maintained
at the National Institute of Health (Porto,
Portugal) in rooms with controlled
temperature (22 ± 2ºC) and humidity (55%
± 10%) and continuous air renovation.
Animals were housed in a 12 h light/12 h
dark cycle (8 am-8 pm). All animal
experiments were performed in accordance
with the National (DL 129/92; DL 197/96; P
1131/97) and European Convention for the
Protection of Animals used for Experimental
and Other Scientific Purposes and related
European Legislation (OJ L 222, 24.8.1999).
Experimental Infections
Urine was collected from S. haematobium-
infected individuals. The individuals were
living in Angola, an endemic area for
schistosomiasis. Informed consent from
patients was obtained. S. haematobium
infection was detected by microscopically
observation of the eggs in the sediment of
centrifuged urine. The eggs were hatched
and with the resulting miracidia, snails from
susceptible species were infected.
Golden hamsters were experimentally
infected with 100 cercariae by tail
immersion. The control animals consisted of
littermates. The cercariae were obtained by
shedding of snails infected with miracidia.
S. Haematobium Total Antigen (Sh)
Production
Angolan strain of S. haematobium adult
worms were collected by perfusion of the
hepatic portal system of golden hamsters at
seven weeks after infection with 100
cercarie. The worms were suspended in PBS
and then sonicated. The protein extract was
then ultracentrifuged and the protein
concentration was estimated using a micro
BCA protein assay reagent kit.
Experimental Design
Forty mice were randomly divided into six
experimental groups. Groups 1 and 3 (n=10)
were Sh-instilled animals. Groups 2 (n=5)
and 4 (n=5) were control animals with their
bladders instilled with saline only. Groups 1
and 2 were sacrificed 20 weeks after
instillation; group 3 and 4 were sacrificed
40 weeks after instillation. For the duration
of the study, the mice’s state of health was
monitored daily.
For intravesical instillation of the parasite
total antigen, 50 g/ml of Sh was
introduced directly into the urinary
bladders of female mice via the urethra
according to the procedure previously
described (Chin et al, 1996). Briefly, animals
were anesthetized with an intraperitoneal
injection of sodium pentobarbital (40
mg/kg). The bladder was catheterized via
the urethra with a 24 gauge plastic
intravenous cannula and the bladder
mucosa was then traumatized to remove the
umbrella cells of the urothelium by instilling
0.1 ml of 0.1N HCl solution for 15 seconds,
Advances in Cancer: Research & Treatment 4
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Mónica C Botelho, Isabel Veiga, Paula A Oliveira, Carlos Lopes, Manuel Teixeira, José M Correia da Costa and
José C Machado (2013), Advances in Cancer: Research & Treatment, DOI: 10.5171/2013.876585
neutralized with 0.1 ml of 0.1N KOH and
flushed with sterile saline.
A total of 150 l of Sh with a concentration
of 50 g/ml was instilled via the cannula.
The dwell time after instillation was
approximately 1 hour. The mice were
turned 90 degrees every 15 minutes to
facilitate whole bladder exposure to the Sh
intravesically instilled. One hour after the
instillation, micturition of the mice was
induced by light abdominal massage so that
the duration of exposure was constant. Body
temperature was maintained with a
homoeothermic bandage. All the
experiments described herein were carried
out under aseptic conditions and performed
once during this study.
All animals were sacrificed by
intraperitoneal administration of sodium
pentobarbital (130 mg/Kg). Complete
necropsies were carefully conducted. All
organs were examined macroscopically for
any changes. The urinary bladders were
inflated in situ by injection of 10%
phosphate buffered formalin (150μl),
ligated around the neck to maintain proper
distension and then were immersed in the
same solution for 12 hours. After fixation,
the formalin was removed; the urinary
bladder was cut into two strips, and
routinely processed. Tissue sections (2 μm)
from urinary bladder, spleen, liver and lung
were stained with haematoxylin and eosin
(HE) to characterize changes and
inflammatory infiltrate.
Mutation Analysis of KRAS Gene
Whole tissue of the bladders was used for
mutational analysis of KRAS codon 12.
Direct sequencing was performed and Big
Dye Terminator (version 1.1) was used
according to instructions of supplier
(Applied Biosystems).
Results
Animals’ General Aspect
During the experimental work no animal
died and all, animals exhibited normal cage
activity. The weight of the animals remained
normal throughout the experiment (data
not shown).
Macroscopic and Microscopic Evaluation
The bladders from all groups showed no
gross lesions. No macroscopic changes were
seen in the liver, lungs or kidneys of all
animals. The mean liver, kidney and bladder
weights, as well as relative liver, kidney and
bladder weights at the end of the study did
not show differences (data not shown).
The bladder histopathological findings
observed in all groups are given in Table 1
and Figure 1. Bladder histopathology of
animals instilled with Sh was characterized
by extensive diffuse or nodular infiltrate
composed predominantly of lymphocytes
admixed with variable number of
eosinophils, and by absence of umbrella
cells, nodular hyperplasia and dysplasia.
The nodular hyperplasia exhibiting budlike
proliferated urothelial cells extending into
the lamina propria. After 20 weeks of
treatment 30% of these animals displayed
urothelial dysplasia a LGIUN (Low Grade
Intra Urothelial Neoplasia), whereas after
40 weeks of treatment we found 70% of the
bladders with this LGIUN, a non-invasive
malignant flat lesion in the urothelium of
these animals. Mastocytes were only found
in the bladders of animals sacrificed after 40
weeks. No bladder histopathological
changes were observed in animals from
control groups. No microscopic changes
were seen in the spleen, lung, kidney and
liver in samples obtained from all animals.
5 Advances in Cancer: Research & Treatment
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Mónica C Botelho, Isabel Veiga, Paula A Oliveira, Carlos Lopes, Manuel Teixeira, José M Correia da Costa and
José C Machado (2013), Advances in Cancer: Research & Treatment, DOI: 10.5171/2013.876585
Table 1: Histopathological Findings in the Sh Instilled Bladders and Controls
Group n W N U I L M D
Group 1
(Sh treated)
10 20 7/10
(70%)
5/10
(50%)
9/10
(90%)
9/10
(90%)
0/10
(0%)
3/10
(30%)
Group 2
(Control)
10 20 10/10
(100%)
9/10
(90%)
2/10
(20%)
2/10
(20%)
0/10
(0%)
0/10
(0%)
Group 3
(Sh treated)
10 40 0/10
(0%)
0/10
(0%)
10/10
(100%)
9/10
(90%)
4/10
(40%)
7/10a
(70%)
Group 4
(Control)
10 40 10/10
(100%)
10/10
(100%)
3/10
(30%)
3/10
(30%)
0/10
(0%)
0/10
(0%) n, number of animals; W, weeks between treatment and sacrifice; N, normal urothelium; U, presence of umbrella cells; I,
inflammatory infiltrate; L, infiltration of lymphocyte; M, infiltration of mastocytes; D, dysplasia. a (p=0.001, Group 3 Vs.
Group 4) (12).
Figure 1: (A and B) Control, (C) Urothelium Displaying an Inflammatory Nodule and a
Lesion with Characteristics of Nodular Hyperplasia, (D) Denudation and Dysplasia, (E)
Inflammatory Infiltrate and Dysplasia, (F) Inflammatory Nodule and Dysplasia (12)
Advances in Cancer: Research & Treatment 6
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Mónica C Botelho, Isabel Veiga, Paula A Oliveira, Carlos Lopes, Manuel Teixeira, José M Correia da Costa and
José C Machado (2013), Advances in Cancer: Research & Treatment, DOI: 10.5171/2013.876585
Frequency of KRAS Mutations in Codon
12
Two (out of 10) of the bladders with
dysplasia presented a KRAS mutation in
codon 12 of exon 2 (Fig. 2). This results in
Gly12Asp mutation. The controls showed no
mutation. We concluded from these results
that the parasite extract of S. haematobium
has carcinogenic ability possibly through
oncogenic mutation of KRAS gene.
Figure 2: KRAS Exon 2 Sequencing Results. (A) Control, (B) Dysplastic Bladder with a
Mutation at Codon 12 (Resulting in a Gly12Asp Mutation).
Discussion
Bladder cancer is a complex disease, with a
multitude of genetic aberrations already
uncovered, and likely more still to come. We
previously demonstrated that Schistosoma
haematobium total antigen has a
carcinogenic ability in a CD-1 mice model.
We showed that intravesically
administration of Sh gave rise to a high
incidence of urothelial dysplasia, a non-
invasive malignant flat lesion, in 70% of the
tested subjects after 40 weeks of treatment.
In the present study, we were able to
investigate this effect further and show that
20% of the dysplastic bladders presented a
mutation in codon 12 of KRAS gene.
It is generally recognized that the
pathogenesis of malignancy involves three
distinct and sequential effects on the target
cells; initiation, promotion and progression.
The first stage is thought to involve the
formation of damage in the DNA resulting in
a somatic cell mutation. The essential
feature of promotion, however, is the
induction of proliferation in the initiated
cells by agents that are not necessarily
carcinogenic per se (Gutiérrez and
Salsamendi, 2001). The sequence of lesions
identified, via histopathology, between
initiation and promotion are designated as
preneoplastic lesions and/or benign
neoplasias (Gutiérrez and Salsamendi,
2001). Their transformation into malignant
lesions is the last of the stages of
carcinogenesis and is the most extended – it
is labelled progression (Klaunig et al, 2000;
Williams, 2001). We cannot state at this
point that KRAS mutation that we detected
in dysplastic bladders induced by Sh
treatment is an initiating event. The
presence of this mutation could have one of
two explanations: either the parasite total
antigen has carcinogenic molecule(s)
sufficiently genotoxic and, consistent with
the mutation observed, caused direct attack
on the RAS gene leading to uncontrolled
proliferation of the cells and causing
dysplasia in the bladders treated with the
parasite antigen; or the parasite antigens
are only mitogenic for the urothelial cells
causing increased proliferation that resulted
in the KRAS mutation via non-carcinogen-
mediated pathways.
In agreement with the first explanation
other researchers have well characterized
7 Advances in Cancer: Research & Treatment
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Mónica C Botelho, Isabel Veiga, Paula A Oliveira, Carlos Lopes, Manuel Teixeira, José M Correia da Costa and
José C Machado (2013), Advances in Cancer: Research & Treatment, DOI: 10.5171/2013.876585
the phenomenon of RAS oncogene activation
in a number of systems. In some cases, such
as the mouse and rat mammary gland, RAS
activation is clearly an important initiating
event, and when tumors are induced by
genotoxic carcinogens the mutations
observed are consistent with direct attack
by the carcinogen on the RAS gene (Stanley,
1995). Also the majority of evidence taken
from several animal studies suggests that
RAS proto-oncogene activation is an early
event. This presumption is derived from
detection of RAS activation in benign (early)
tumors of skin, lung and liver. Based upon
tumor induction following a brief exposure
to carcinogens that produce a chemical-
specific point mutation in RAS activation,
researchers have suggested that activation
of RAS may be the initiating event in some
model systems (Moronpot et al, 1995).
Furthermore, the activated RAS gene has
been detected in many benign tumors,
including mouse skin papillomas, mouse
lung and liver adenomas, and basal cell and
clitoral gland tumors of the rat. This implies
that the activated RAS was present in the
cell that clonally expanded to these benign
tumors. Thus, activation of the RAS proto-
oncogene may be the initiation event in
some model systems (Stowers et al, 1987).
Few studies were carried out to investigate
the role and frequency of activated RAS in
schistosomiasis-associated bladder cancer
(Badawi, 1996). The mutations were not
detected in two previous studies (Fugita et
al, 1987; Ramchurren N et al, 1995). These
findings indicate that the advanced stage of
schistosomiasis-associated bladder cancer
may not involve a high frequency of
detectable mutations within the RAS genes.
It is known that more than 90% of the SCC
in the schistosome-infected bladder were
advanced (stages T3 and T4) at the time of
diagnosis. However, this does not exclude
the possibility that activation in RAS could
be an important factor in the initiation as
well as promotion and progression of
schistosomiasis-associated bladder cancer
(Badawi, 1996).
Further studies are needed to address the
functional effect of KRAS mutations and the
clinical relevance of these mutations in
schistosomiasis-associated bladder cancer.
Acknowledgements
We would like to express our deepest
appreciation to Mrs. Lígia Lourenço for her
technical assistance, Dr. Paulo Vieira and
Mrs. Maria de Lurdes Delgado for animal
care and antigen production.
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